Cloning And Function Analysis Of Related Genes Involved In Rubber Biosynthesis Of Eucommia Ulmoides

Eucommia Ulmoides Rubber(EUR) is a quality natural rubber resources and has a unique structure and properties. Although EUR can develop a variety of new industrial materials, high cost of production because the low content in leaves and fruit have become EUR industrial development bottlenecks. It is not yet clear that main pathway, regulation mechanism and key enzyme gene of EUR biosynthesis now. Identified the major pathway, critical period and key gene of EUR biosynthesis by analyzing expression differences and subcellular localization of related genes on the based of genome and transcriptome. This research reveals the bioaccumulation process of EUR and provides important theoretical basis for cultivating the high-yielding varieties of EUR. The main results are as follows.1. The research has successfully cloned the genomic and full-length cDNA of EUR biosynthesis. The length of genomic of EuTIDSl-5is6444bp,5063bp,13745bp,6476bp,3412bp and contain11,12,12,11,12exons respectively.Promoter region of EuTIDS1-5contains TATA-box of14%-37%, CAAT-box of36%-54%and light of3-19%. EuTIDS\,3have cis-acting elements of root-specific expression, EuTIDS2,4have specific cis-acting element conferring high transcription levels, EuTIDS4has cis-acting elements of MeJA-responsiveness and gibberellin-responsiveness, EuTIDS5has ethylene-responsive element.2. Identified key gene of EUR biosynthesis. The research of relative genes in leaves and fruit at different time have done by Real-time PCR. The expression of EuTIDS1in leaves is26%-86%more than fruit is not consistent with the EUR content of fruit is1.3-2.0times of leaves at the same time, EuTIDS3are expressed in leaves and fruit, but the expression levels do not change with the EUR content at different time. So EuTIDS1and EuTIDS3have no relationship with the EUR biosynthesis. The expression of EuTIDS5is not highest until pollinate ovary after50days, EUR are rapid accumulation and the growth rate of EUR reach highest at this time. The expression of EuTIDS5in fruit is48%-99%higher than leaves and EUR content of fruit is1.3-2.0times of leaves at the same time. Therefore, EuTIDS5is closely relation to EUR biosynthesis. Constructs of over expression and RNAi for EuTIDS5are generated, sixteen over expression plants are obtained and content of trans-polyisoprenoid is up to0.28%. Compared with wild-type bark, over expression of EuTIDSS is2.5times as high as content of wild-type, but RNAi of EuTIDS5is half content of wild-type.Indicating that the expression of EuTIDS5is beneficial to trans-polyisoprenoid synthesis and is the key gene of EUR biosynthesis.3. Identified the main upstream pathway of EUR biosynthesis. EUR is a polyterpene and terpenes biosynthesis occurs via two different pathways:the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway, respectively. According to the date of transcriptome found that78%of genes (EuACOT8-10、 EuHMGS4-6、EuHMGR12,13,15-17、EuMK3-4、EuPMK3、 EuMDP7-8,10-11) of the MEP pathway in leaves expression are20-90%higher than young fruit, this is not consistent with the EUR content of fruit is1.2-3.2times more than leaves at the same time.75%of genes(EuACOTl-7、EuHMGSl、 EuHMGR2-7,11、 EuMK1-2、EuPMK1-2、EuMDPl-5) of the MVA pathway in fruit expression are40-98%more than leaves, this is consistent with the EUR content of fruit are far higher than leaves at the same time. The study of subcellular localization also show that EuDXR, EuMCT, EuCMK, EuMDS of the MEP pathway co-localizes to the chloroplast, but EUR content of root and bark are as2.5times high as leaves, EuACOT, EuHMGR of the MVA pathway and EuTIDSl-5co-localizes to the endoplasmic reticulum (ER) and this is consistent with EUR content of root and bark are far higher than leaves. So EUR accumulate in the cytoplasm and the MVA pathway is the main way of synthetic EUR.4. Identified critical period of EUR biosynthesis. The growth rate of EUR reach the highes in pollinate ovary after50days. The75%of genes (EuACOTl-6,8-10、 EuHMGSl-6、 EuHMGR2-11-18、EuMK1-4、 EuPMK1-3、 EuMDP1-5,7-8,10-11) of the MVA pathway in young fruit expression are40-98%and34%-99%more than leaves and fruit respectively. EuIPI、 EuMLP、 EuREF、EuSRPP in young fruit expression are39-73%,52%-99%,61-97%and83%more than fruit respectively. At the same time, the expression of EuTIDS5in young fruit and growth rate of EUR are rapidly increased, So critical period of EUR biosynthesis is young fruit growth period.